The present invention relates to the production of biotin using genetically engineered organisms.
Biotin (vitamin B8 or vitamin H) is a nonpolypeptide coenzyme molecule involved in enzyme-catalyzed reactions requiring carboxyl group transfers. Biotin, like many coenzymes, cannot be synthesized by animals and must instead be obtained exogenously from plants or microorganisms in the diet. Large-scale production of biotin for commercial use as a dietary supplement is therefore desirable. Genetically-modified microorganisms may produce dietary supplements in commercially advantageous amounts and the present invention provides an approach to accomplish this goal for biotin.
The synthesis of biotin in microorganisms is achieved by both chemical and fermentation methods. At the genetic level, microbial synthesis of biotin in vivo is driven from an operon containing a cluster of genes. The arrangement of genes within the cluster is specific to each bacterial species. Additionally, the concentration of intermediates and product, as well as the amount of biotinylated protein in a cell, regulates biotin operon transcriptional activity (Weaver et al., 2001). For instance, repression of the operon occurs through binding of the birA gene product together with biotinoyl-AMP to the regulatory sequence that lies between the bioA gene and the biotin operon (Weaver et al., 2001; Brown and Kamogawa, 1991).
Biotin synthesis requires the proteins encoded by the bioH, bioC, bioF, bioA, bioD, and bioB genes. (Otsuka A J et al., 1988; Sakurai N et al., 1996; Brown and Kamogawa, 1991; Pollock and Barber, 2001; Picciocchi et al., 2001; Ploux and Marquet, 1992; Gibson et al. 1995). The enzymatic steps involved in the biotin synthetic pathway from pimelic acid to biotin has not been elucidated fully in Pseudomonas. Generally, however, as shown in FIG. 1, the steps are predicted to include converting (1) pimelic acid to pimelyl-CoA (PmCoA) by the bioC gene product, which is unidentified, and pimeloyl CoA synthetase that is encoded by bioH (Ploux et al., 1992); (2) Pimelyl-CoA to 7-keto-8-amino pelargonic acid (KAPA) by 7-KAP synthetase (BioF); (3) KAPA to 7,8-diamino-pelargonic acid (DAPA) by DAPA aminotransferase (BioA); (4) DAPA to dethiobiotin by dethiobiotin synthetase (BioD); and (5) dethiobiotin to biotin by biotin synthetase (BioB). See FIG. 1. Synthesis of PmCoA reportedly involves different enzymatic steps in different microorganisms. (Bower et al., 1996)
The biotin operon for E. coli consists of a 5.8 Kb region containing five biotin operon genes, bioA, bioB, bioF, bioC and bioD (Otsuka et al., 1988). The bioA gene runs in the opposite direction with control of the operon being between the bioA and bioB sequences, basepairs 807,191 through 812,170 for E. coli K12 (NCBI accession number: NC—000913). The regulatory region is shared by bioA and the rest of the cassette having two promoters running in the opposite direction and on either side of the operator (Brown and Kamogawa, 1991). BioH, in E. coli, is located several kilobases downstream from the operon starting at nucleotide 3,542,096. This gene arrangement for the biotin operon is similar in Serratia marcescens (Sakurai et al., 1996). In Bacillus subtilis, the arrangement is W(H)AFDBIorf2 (Bower et al., 1996). Similarly, Pseudomonas aeruginosa has the bioH as part of the biotin operon but it is unknown whether mutabilis similarly does. Additionally, the bioA gene in P. aeruginosa is further away from the biotin BFHCD operon by close to 100 Kb but within the operon for B. subtilis. Although similar gene products are necessary for synthesis of biotin in these different bacteria, the arrangement of the genes encoding the necessary proteins varies from genus to genus. (Rodionov et al., 2002)
Previously genetically modified microorganisms have suffered from poor conversion of dethiobiotin to biotin, inefficient promoters, poor gene cluster arrangements and unsatisfactory biotin production. The present invention overcomes these deficiencies through the combination of specific genes whose encoded products are involved in biotin synthesis. Furthermore, the present invention discloses the creation of a mutant strain of Pseudomonas mutabilis that produces gram per liter amounts of biotin when transformed with a unique synthetic operon engineered according to the teachings disclosed herein.